兔原始生殖细胞体外培养的研究

doi:10.11843/j.issn.0366-6964.2020.10.012

Abstract

Abstract: The purpose of the present study was to explore the optimal condition for the isolation and culture of rabbit primordial germ cells(PGCs) in vitro, and to establish a mature and stable method for the isolation and culture of rabbit PGCs in vitro. Firstly, trypsin digestion and mechanical methods were used to explore the best method for isolating PGCs from 14- to 18-day-old fetal rabbits in vitro, and trypsin digestion, mechanical method and digestion with the feeder layer were used to explore the optimal way to passage the isolated PGCs. Then, 4 different culture media(A, B, C and D (control)) were used to explore the effect of different concentrations of cytokines on the morphological changes and colony formation of rabbit PGCs. Secondly, rabbit PGCs were identified by alkaline phosphatase staining (AKP), and the expression of transcription factor Oct-4 was detected by real-time PCR (RT-PCR). The results showed that the number of rabbit PGCs colonies isolated by mechanical method was 2.2 times higher than those isolated by trypsin digestion method. Rabbit embryo fibroblasts (REF) were passaged to the second generation (P2) using trypsin digestion method and digestion with the feeder layer method, and REF were passaged to the fourth generation (P4) using the mechanical method. The largest number of colonies and better colony morphology were observed when PGCs were cultured in medium B(basic medium＋10% fetal bovine serum (FBS) ＋2 ng·mL^-1 of transforming growth factor-β1 (TGF-β1) ＋4 ng·mL^-1of basic fibroblast growth factor (bFGF) ＋10 ng·mL^-1leukemia inhibitory factor (LIF)). Rabbit PGCs remained undifferentiated for a longer time when cultured in medium B. AKP staining showed that the isolated PGCs was positive in red and black, and expression of Oct-4 gene was also detected in the isolated PGCs by RT-PCR. The results showed that mechanical isolation method and mechanical passage method were the optimal ways to isolate and passage rabbit primary PGCs, respectively. Supplementation with appropriate concentration of cytokines into culture medium was beneficial for rabbit PGCs to maintain a large number of colonies and undifferentiated state for a longer time in vitro. In the present study, the culture methods of rabbit PGCs in vitro were screened and optimized, which would lay a technical foundation for the further establishment of a stable and mature rabbit PGCs cell line.

Key words: Oryctolagus cuniculus, primordial germ cells (PGCs), isolation methods, passage methods, cytokines

CLC Number:

S829.1

Lü Haimiao, ZHU Hanyu, PENG Zhan, YAN Chenbo, YANG Dexin, HU Wenju, DING Xuefen, WANG Xinzhuang. Study on the Culture of Rabbit (Oryctolagus cuniculus) Primordial Germ Cells in Vitro[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2443-2452.

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Study on the Culture of Rabbit (Oryctolagus cuniculus) Primordial Germ Cells in Vitro

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